Table 1.
Potential genomic targets of PEG3 in the mouse genome (mm9).
Fig 1.
DNA-binding affinity of two sets of competitors.
The genomic regions identified through ChIP-Seq were examined to identify potential DNA-binding sites for PEG3 with its known core motif, 5’-TGGC-3’, which is marked in red. These selected regions were competed against the 32P-labeled Pgm2l1 oligonucleotide duplex. These assays derived two types of sequences: the competitors with high (A) and low (B) affinity. The labels for the pictures are as follow: Neg, a negative control without nuclear extract; Pos, a positive control with nuclear extract. The gene names in the remaining lanes indicate the oligonucleotide duplexes that have been tested as competitors at 100x molar ratio to the Pgm2l1 probe.
Fig 2.
Comparison of the competitors with high and low affinity.
(A) The competitors with high and low affinity were individually analyzed using the MEME program. This motif search derived two similar but slightly different motifs: GTGGCAGT and TGGCACnC. (B) Two individual competitors with high affinity were further analyzed with their mutated versions of competitors. The GG-to-AA mutation is marked in blue whereas the T-to-C mutation is marked with red. The competition was performed with each duplex against the Pgm2l1 probe at the molar ratio of 100 to 1.
Fig 3.
Consensus DNA-binding motif of PEG3.
(A) The newly derived DNA-binding motif is presented with a tabulated matrix. This matrix has been derived from the sequence alignment of 12 competitors with high affinity shown in (B). The new motif was also compared with the previously defined motif of PEG3 using the sequence of the Pgm2l1 oligonucleotide duplex (C). Two separate motifs localized in an opposite direction are indicated with two arrows.
Fig 4.
Genomic locus containing Slc38a2 and Slc38a4.
(A) The 500-kb genomic region containing solute carrier family 38 in mouse chromosome 15 is presented with the image derived from the UCSC genome browser. Small vertical lines indicate the exons of each gene. (B) The magnified view of Slc38a2 is presented with histone modification profiles. The promoter and 5’-side enhancer of Slc38a2 are modified with high levels of H3K4me1, H3K4me3 and H3K27ac. These two regions have also been identified through PEG3 ChIP-Seq as indicated by the positions of three ChIP-Seq peaks.
Fig 5.
ChIP and qRT-PCR analyses on Slc38a2 and Slc38a4.
(A) Individual ChIP experiments were performed to further confirm in vivo binding of PEG3 to several candidate regions using the chromatin prepared from neonatal brains. The immunoprecipitated DNA was used as templates for PCR amplification. The results of these ChIP experiments were presented in the following order: Input, Neg and PEG3 IP. Each tested locus was indicated with its gene name on left. (B) RT-PCR analyses using the total RNA isolated from the brains and hearts of wild-type (WT) and knockout (KO) littermates for Peg3. The observed changes in the expression levels of Slc38a2 and Slc38a4 were further analyzed using qRT-PCR (C). For this series of analyses, we used the β-actin as an internal control.
Fig 6.
The paternal allele-specific expression of Slc38a4 was tested using the F1 hybrid derived from the interspecific crossing of a male C57BL/6J (B6) heterozygous for Peg3 KO and a female PWD/PhJ breeder. The differentiation of two parental alleles was visualized through a restriction enzyme (PvuII) digestion on the RT-PCR products that had been derived from the transcribed region of Slc38a4. The schematic representation is shown on the bottom panel. The paternal allele-specific expression was detected in both WT and KO, indicating that the observed up-regulation of Slc38a4 in Peg3 KO involves only the active paternal allele, but not the imprinted maternal allele.